Abstract Exposure to polycyclic aromatic hydrocarbons (PAHs) has been associated with risk of bladder cancer and with increased bulky DNA adduct levels in several studies, mainly in smokers. We investigated the relation between bulky PAH-DNA adducts in peripheral blood mononuclear cells and bladder cancer in nonsmoking subjects from a large hospital-based case-control study in Spain. Additionally, we examined the association between DNA adduct formation and several air pollution proxies. The study comprised 76 nonsmoking cases and 76 individually matched controls by sex, region of residence, age, and smoking status (never, former). To maximize the relevance of the DNA adduct measurement as a proxy of PAH exposure, subjects selected had not changed residence, occupation, and major lifestyle factors during the last 10 years. Bulky DNA adducts were measured using the 32P-postlabeling technique, nuclease P1 treatment. The percentage of detectable adducts was higher in controls (41%) than in cases (32%) with an odds ratio of 0.75 (95% confidence interval, 0.36-1.58). In an analysis limited to controls, a higher percentage of DNA adducts was found among those whose last residence was in a big city (50%) compared with those living in villages (19%; P = 0.04). No consistent associations were found for other markers of air pollution. In this study, among nonsmokers with stable environmental and lifestyle factors, bulky DNA adducts were not associated with bladder cancer risk. Results do not support an association of bladder cancer risk with low-level exposure to PAHs as measured through the formation of bulky DNA adducts in peripheral mononuclear cells. (Cancer Epidemiol Biomarkers Prev 2007;16(10):2155–9)